ARF6 regulates a plasma membrane pool of phosphatidylinositol(4,5)bisphosphate required for regulated exocytosis

ADP-ribosylation factor (ARF) 6 regulates endosomal plasma membrane trafficking in many cell types, but is also suggested to play a role in Ca²⁺-dependent dense-core vesicle (DCV) exocytosis in neuroendocrine cells. In the present work, expression of the constitutively active GTPase-defective ARF6^Q67L mutant in PC12 cells was found to inhibit Ca²⁺-dependent DCV exocytosis. The inhibition of exocytosis was accompanied by accumulation of ARF^Q67L, phosphatidylinositol 4,5-bisphosphate (PIP₂), and the phosphatidylinositol 4-phosphate 5-kinase type I (PIP5KI) on endosomal membranes with their corresponding depletion from the plasma membrane. That the depletion of PIP₂ and PIP5K from the plasma membrane caused the inhibition of DCV exocytosis was demonstrated directly in permeable cell reconstitution studies in which overexpression or addition of PIP5KIγ restored Ca²⁺-dependent exocytosis. The restoration of exocytosis in ARF6^Q67L-expressing permeable cells unexpectedly exhibited a Ca²⁺ dependence, which was attributed to the dephosphorylation and activation of PIP5K. Increased Ca²⁺ and dephosphorylation stimulated the association of PIP5KIγ with ARF6. The results reveal a mechanism by which Ca²⁺ influx promotes increased ARF6-dependent synthesis of PIP₂. We conclude that ARF6 plays a role in Ca²⁺-dependent DCV exocytosis by regulating the activity of PIP5K for the synthesis of an essential plasma membrane pool of PIP₂.